Signaling Pathways in Rheumatoid Arthritis

Due to unique cellular responses observed in HFLS-RA cells (compared to HFLS-OA or HFLS control cells), cell signaling pathways were examined only in HFLS-RA cells. Several signal transduction pathways involved in the transformation of HFLS-RA cells to an aggressive phenotype were examined (16 (link), 50 (link), 59 (link)). Briefly, HFLS cells were incubated with antigens for 15-min, 30-min, 1-h and 4-h, cellular lysates were prepared as above, and the following primary antibodies (all polyclonal rabbit IgGs) were utilized to probe the samples; anti-β-actin antibody (endogenous control) (Novus Biologicals), anti-SAPK/JNK (#9252), anti-phospho-SAPK/JNK (Thr183/Tyr185) (#9251), anti-Erk1/2 (p44/42 MAPK) (#9102), anti-phospho-Erk1/2 (p44/42 MAPK; Thr202/Tyr204) (#9101), anti-Akt (#9272), anti-phospho-Akt (Ser473) (#9271), anti-p38 MAPK (#9212), anti-phospho-p38 MAPK (Thr180/Tyr182) (#4511), anti-Paxillin (#2542), anti-phospho-Paxillin (Tyr118) (#2541), anti-FAK (#3285), anti-phospho-FAK (Tyr397) (#3283) (Cell Signaling Technology, Danvers, MA, USA). HRP-conjugated anti-rabbit IgG (Jackson ImmunoResearch) was used as a secondary antibody. Respective band intensities were measured and reported as outlined above. The maximum and minimum activation of signaling pathways for HFLS lysates was determined based on positive (treatment with LPS) and negative (treatment with media) controls.

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Aripova N., Duryee M.J., England B.R., Hunter C.D., Mordeson J.E., Ryan E.M., Daubach E.C., Romberger D.J., Thiele G.M, & Mikuls T.R. (2023). Citrullinated and malondialdehyde-acetaldehyde modified fibrinogen activates macrophages and promotes an aggressive synovial fibroblast phenotype in patients with rheumatoid arthritis. Frontiers in Immunology, 14, 1203548.

Publication 2023

anti-Erk1/2 (p44/42 MAPK) anti-phospho-Erk1/2 ( anti-Akt anti-phospho-Akt (Ser473) anti-p38 MAPK anti-phospho-p38 MAPK anti-Paxillin anti-phospho-Paxillin (Tyr118) anti-FAK anti-phospho-FAK (Tyr397) HRP-conjugated anti-rabbit IgG

Actin Anti antibody Anti igg Antibodies Antibody Antigens Biologicals Cellular Novus P38 mapk P44 mapk Paxillin Phenotype Rabbit Signaling pathways

Corresponding Organization : VA Nebraska Western Iowa Health Care System

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Variable analysis

independent variables

Antigen incubation time (15-min, 30-min, 1-h, 4-h)

dependent variables

Activation of signaling pathways (SAPK/JNK, Erk1/2, Akt, p38 MAPK, Paxillin, FAK) measured by phosphorylation levels

control variables

HFLS-RA cell line
β-actin as endogenous control

controls

Positive control: Treatment with LPS
Negative control: Treatment with media

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